This invention relates to nucleic acids and encoded polypeptides which interact with the TGF-xcex2 receptor complex and which is a negative regulator of TGF-xcex2 signaling. The invention also relates to agents which bind the nucleic acids or polypeptides. The invention further relates to methods of using such nucleic acids and polypeptides in the treatment and/or diagnosis of disease.
During mammalian embryogenesis and adult tissue homeostasis transforming growth factor xcex2 (TGF-xcex2) performs pivotal tasks in intercellular communication (Roberts et al., Growth Factors, 8:1-9, 1993). The cellular effects of theis pleiotropic factor are exerted by ligand-induced hetero-oligomerization of two distantly related type I and type II serine/threonine kinase receptors, Txcex2R-I and Txcex2R-II, respectively (Lin and Lodish, Trends Cell Biol., 11:972-978.,1993; Derynck, Trends Biochem. Sci., 19:548-553, 1994; Massague and Weis-Garcia, Cancer Surv. 27:41-64, 1996; ten Dijke etal., Curr. Opin. Cell Biol., 8:139-145, 1996). The two receptors, which both are required for signaling, act in sequence; Txcex2R-I is a substrate for the constitutively active Txcex2R-II kinase (Wrana et al., Nature, 370:341-347, 1994; Wieser et al., EMBO J., 14:2199-2208, 1995). TGF-xcex2 forms part of a large family of structurally related proteins which include activins and bone morphogenetic proteins (BMPs) that signal in a similar fashion, each employing distinct complexes of type I and type II serine/threonine kinase receptors (Lin and Lodish, 1993; Derynck, 1994; Massague and Weis-Garcia, 1996; ten Dijke et al., 1996).
Genetic studies of TGF-xcex2-like signaling pathways in Drosophila and Caenorhabditis elegans have led to the identification of mothers against dpp (Mad) (Sekelsky et al., Genetics, 139:1347-1358, 1995) and sma (Savage etal., Proc. Natl. Acad. Sci. USA, 93:790-794, 1996) genes, respectively. The products of these related genes perform essential functions downstream of TGF-xcex2-like ligands acting via serine/threonine kinase receptors in these organisms (Wiersdorf et al., Development, 122:2153-2163,1996; Newfeld et al., Development, 122:2099-2108, 1996; Hoodless et al., Cell, 85:489-500, 1996). Vertebrate homologs of Mad and sma have been termed Smads (Derynck et a., Cell, 87:173, 1996) or MADR genes (Wrana and Attisano, Trends Genet., 12:493-496, 1996). Genetic alterations in Smad2 and Smad4/DPC4 have been found in specific tumor subsets, and thus Smads may function as tumor suppressor genes (Hahn et al., Science, 271:350-353, 1996; Riggins et al., Nature Genet., 13:347-349, 1996; Eppert et al., Cell, 86:543-552, 1996). Smad proteins share two regions of high similarity, termed MH1 and MH2 domains, connected with a variable proline-rich sequence (Massague, Cell 85:947-950, 1996; Derynck and Zhang, Curr. Biol., 6:1226-1229, 1996). The C-terminal part of Smad2, when fused to a heterologous DNA-binding domain, was found to have transcriptional activity (Liu et al., Nature, 381:620-623, 1996; Meersseman et al., Mech. Dev., 61:127-140, 1997). The intact Smad2 protein when fused to a DNA-binding domain, was latent, but transcriptional activity was unmasked after stimulation with ligand (Liu et al., 1996).
Different Smads specify different responses using functional assays in Xenopus. Whereas Smadl induces ventral mesoderm, a BMP-like response, Smad2 induces dorsal mesoderm, an activin/TGF-xcex2-like response (Graff et al., Cell, 85:479-487, 1996; Baker and Harland, Genes and Dev., 10:1880-1889 1996; Thomsen, Development, 122:2359-2366, 1996). Upon ligand stimulation Smads become phosphorylated on serine and threonine residues; BMP stimulates Smadl phosphorylation, whereas TGF-xcex2 induces Smad2 and Smad3 phosphorylation (Hoodless et al., 1996; Liu et al., 1996; Eppert et al., 1996; Lechleider et al., J Biol. Chem., 271:17617-17620, 1996; Yingling et al., Proc. Natl. Acad. Sci. USA, 93:8940-8944, 1996; Zhang et al., Nature, 383:168-172, 1996; Macias-Silva et al., Cell, 87:1215-1224, 1996; Nakao et al., J. Biol. Chem., 272:2896-2900, 1996).
Smad4 is a common component of TGF-xcex2, activin and BMP signaling (Lagna et al., Nature, 383:832-836, 1996; Zhang et al., Curr. Biol., 7:270-276, 1997; de Winter et al., Oncogene, 14:1891-1900, 1997). Smad4 phosphorylation has thus far been reported only after activin stimulation of transfected cells (Lagna et al., 1996). After stimulation with TGF-xcex2 or activin Smad4 interacts with Smad2 or Smad3, and upon BMP challenge a heteromeric complex of Smad4 and Smad I has been observed (Lagna et al., 1996). Upon ligand stimulation, Smad complexes translocate from the cytoplasm to the nucleus (Hoodless et al., 1996; Liu et al., 1996; Baker and Harland, 1996; Macias-Silva et aL., 1996), where they, in combination with DNA-binding proteins, may regulate gene transcription (Chen et al., Nature, 383:691-696, 1996).
The invention provides isolated Smad2 polypeptides and agents which bind such polypeptides, including antibodies. The invention also provides isolated nucleic acid molecules which encode the foregoing polypeptides, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The foregoing can be used in the diagnosis or treatment of conditions characterized by TGF-xcex2 signal transduction. The invention also provides methods for identifying pharmacological agents useful in the diagnosis or treatment of such conditions. Here, the identification of Smad2 amino acid residues phosphorylated in vivo is reported.
According to one aspect of the invention, an isolated Smad2 polypeptide is provided. The polypeptide has the amino acid sequence of SEQ ID NO:2 or its human homolog except that the polypeptide includes a mutation comprising a non-serine amino acid located at one or more of amino acids 464, 465 and 467. In certain embodiments, the isolated Smad2 polypeptide compises a mutation which is located at a position or positions selected from the group consisting of 464; 465; 467; 464 and 465; 464 and 467; 465 and 467; and 464, 465 and 467. Preferably the isolated Smad2 polypeptide comprises a mutation or mutations of the serine residues to alanine residues (e.g., Ser465A) or aspartic acid residues (e.g., Ser465D) such as those selected from the group consisting of Ser464A; Ser465A; Ser467A; Ser464A and Ser465A; Ser464A and Ser467A; Ser465A and Ser467A; Ser464A, Ser465A and Ser465A; Ser465D; Ser467D; and Ser465D and Ser467D.
In other embodiments, the foregoing isolated polypeptide consists of a fragment or variant of the foregoing which retains the activity of the foregoing.
According to still another aspect of the invention, nucleic acid molecules which encode the foregoing polypeptides are provided. The nucleic acids can be composed of natural and/or non-natural nucleotides and linked with natural and/or non-natural internucleoside bonds.
According to still another aspect of the invention, the invention involves expression vectors, and host cells transformed or transfected with such expression vectors, comprising the nucleic acid molecules described above.
According to another aspect of the invention, there are provided isolated Smad4 binding polypeptides comprising the amino acid sequence of SEQ ID NO:3, which selectively bind a Smad4 protein or fragment thereof, provided that the isolated polypeptide is not wild type Smad2. In certain embodiments, the isolated Smad4 binding polypeptide comprises the C-terminal 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20amino acids of SEQ ID NO:2. Preferably, the isolated Smad4 binding polypeptide of comprises the amino acid sequence of SEQ ID NO:4. In other preferred embodiments, the foregoing isolated Smad4 binding polypeptides are phosphorylated on one or more amino acids selected from the group consisting of Ser464, Ser465, Ser467, Ser464/Ser465, Ser464/Ser467, Ser465/Ser467 and Ser464/Ser465/Ser467.
According to yet another aspect of the invention, the invention involves an isolated polypeptide which binds selectively any of the foregoing isolated polypeptides, provided that the isolated polypeptide is not Txcex2R-I, Txcex2R-II or Smad4. In preferred embodiments, the isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to the polypeptides of the invention). Particularly preferred antibodies include monoclonal antibodies.
In another aspect of the invention, an isolated polypeptide is provided which binds selectively to at least one pathway-restricted Smad polypeptide or fragment thereof having the C-terminal amino acid sequence of SEQ ID NO:5. The pathway-restricted Smad polypeptide or fragment thereof is phosphorylated on at least one serine residue of SEQ ID NO:5, and the isolated polypeptide is not Txcex2R-I, Txcex2R-II or Smad4. In certain embodiments, the pathway restricted Smad polypeptide is selected from the group consisting of Smad1, Smad3, Smad5 and Smad9. In other embodiments, the at least one serine residue is selected from the group consisting of the second serine residue, the third serine residue and the second and third serine residues. In preferred embodiments, the isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to pathway-restricted Smad polypeptides). Particularly preferred antibodies include monoclonal antibodies. In certain embodiments, the antibody or antibody fragment binds selectively to one of the pathway restricted Smad polypeptides.
According to still another aspect of the invention, methods for inhibiting TGF-xcex2 signal transduction in a mammalian cell are provided. The methods involve contacting a mammalian cell with an amount of an inhibitor of phosphorylation of endogenous Smad2 effective to reduce TGF-xcex2 signal transduction in the mammalian cell. In certain embodiments of the foregoing methods, the inhibitor is a dominant negative Smad2 polypeptide, such as the foregoing Smad2 polypeptides which include a mutation at one or more of residues 464, 465 and 467.
The invention in still another aspect provides compositions comprising a Smad2 polypeptide which includes a mutation at one or more of residues 464, 465 and 467, and a pharmaceutically acceptable carrier.
The invention in a further aspect involves a method for decreasing TGF-xcex2 signal transduction activity in a subject. An agent that selectively binds to a TGF-xcex2 receptor and blocks TGF-xcex2 signaling is administered to a subject in need of such treatment, in an amount effective to decrease Smad2 TGF-xcex2 signal transduction activity in the subject. Preferred agents are Smad2 polypeptides which include a mutation at one or more of serines 464, 465 and 467, particularly those which are substituted with alanines or aspartic acids.
According to another aspect of the invention, methods are provided for identifying lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated with Smad2/TGF-xcex2 receptor interaction. The methods involve forming a mixture of a Smad2 polypeptide comprising a mutation at one or more of serines 464, 465 and 467, a TGF-xcex2 receptor, and a candidate pharmacological agent. The mixture is incubated under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of specific binding of the TGF-xcex2 receptor by the Smad2 polypeptide. A test amount of the specific binding of the TGF-xcex2 receptor by the Smad2 polypeptide then is detected. Detection of a reduction in the foregoing activity in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which disrupts the Smad2/TGF-xcex2 receptor interaction. Detection of an increase in the foregoing activities in the presence of the candidate pharmacological agent indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which enhances Smad2/TGF-xcex2 receptor interaction. Preferably the Smad2 polypeptide comprises a mutation or mutations selected from the group consisting of Ser464A; Ser465A; Ser467A; Ser464A and Ser465A; Ser464A and Ser467A; Ser465A and Ser467A; Ser464A, Ser465A and Ser465A; Ser465D; Ser467D; and Ser465D and Ser467D.
According to a further aspect of the invention, methods are provided for identifying lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated with TGF-xcex2-mediated Smad2 signal transduction activity. The methods involve forming a mixture of a wild type Smad2 polypeptide, a TGF-xcex2 receptor, and a candidate pharmacological agent comprising a mutated Smad2 polypeptide, preferably having a mutation at one or more of serines 464, 465 and 467. The mixture is incubated under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of TGF-xcex2 mediated phosphorylation of the wild type Smad2 polypeptide. A test amount of TGF-xcex2 mediated phosphorylation of the wild type Smad2 polypeptide then is detected. Detection of a reduction in the test amount of TGF-xcex2 mediated phosphorylation of the wild type Smad2 polypeptide relative to the first amount of TGF-xcex2 mediated phosphorylation of the wild type Smad2 polypeptide indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which disrupts the TGF-xcex2 mediated Smad2 signal transduction activity. Preferably the mutated Smad2 polypeptide comprises a mutation or mutations selected from the group consisting of Ser464A;
Ser465A; Ser467A; Ser464A and Ser465A; Ser464A and Ser467A; Ser46SA and Ser467A;
Ser464A, Ser465A and Ser465A; Ser465D; Ser467D; and Ser465D and Ser467D, or TGF-xcex2 receptor binding fragments thereof.
According to another aspect of the invention, methods for determining the amount of a pathway-restricted Smad polypeptide having a phosphorylated C-terminal serine residue in a biological sample are provided. The methods include contacting the biological sample with an isolated polypeptide that selectively binds at least one phosphorylated serine of the amino acid sequence as set forth in SEQ ID NO:5, determining the binding of the isolated polypeptide to the pathway-restricted Smad polypeptide, and comparing the binding to a control as a determination of the amount of a pathway-restricted Smad polypeptide having a phosphorylated C-terminal serine residue in the biological sample. The biological sample can be a biological extract such as in vitro extract, or can be tissue sample for in vivo or in vitro immunohistochemistry analysis. In certain embodiments, the pathway-restricted Smad polypeptide is selected from the group consisting of Smad1, Smad2, Smad3, SmadS, and Smad9. In preferred embodiments, the isolated binding polypeptides include antibodies and fragments of antibodies, including monoclonal antibodies. In certain of the preferred embodiments, the antibody or antibody fragment selectively binds a pathway-restricted Smad polypeptide having at least one phosphorylated C-terminal serine residue selected from the group consisting of the second serine residue of SEQ ID NO:5, the third serine residue of SEQ ID NO:5 and the second and third serine residues of SEQ ID NO:5.
Accordig to yet another aspect of the invention, methods for identifying lead compounds for a pharmacological agent which modulate phosphorylation of the C-terminal serine residues of a pathway-restricted Smad polypeptide are provided. The methods include forming a mixture comprising a pathway-restricted Smad polypeptide having at least one C-terminal serine residue, a TGF-xcex2 superfamily receptor or receptor complex capable of phosphorylating the at least one C-terminal serine residue, and a candidate pharmacological agent. The mixture is incubated under conditions which, in the absence of the candidate pharmacological agent, permit a first amount of phosphorylation of the at least one C-terminal serine residue by the TGF-xcex2 superfamily receptor or receptor complex. A test amount of the phosphorylation of the at least one C-terminal serine residue by the TGF-xcex2 superfamily receptor or receptor complex then is detected. A reduction of the test amount of phosphorylation relative to the first amount of phosphorylation indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which reduces phosphorylation of the at least one C-terminal serine residue of a pathway-restricted Smad polypeptide. An increase of the test amount of phosphorylation relative to the first amount of phosphorylation indicates that the candidate pharmacological agent is a lead compound for a pharmacological agent which increases the phosphorylation of the C-terminal serine residues of a pathway-restricted Smad polypeptide. In certain embodiments, the pathway-restricted Smad polypeptide is selected from the group consisting of Smad1, Smad2, Smad3, Smad5, and Smad9. In other embodiments, the at least one C-terminal serine residue is selected from the group consisting of the second serine residue of SEQ ID NO:5, the third serine residue of SEQ ID NO:5 and the second and third serine residues of SEQ ID NO:5. In still other embodiments, the step of forming a mixture comprising a pathway-restricted Smad polypeptide, a TGF-xcex2 superfamily receptor or receptor complex, and a candidate pharmacological agent includes contacting a cell which includes a pathway-restricted Smad polypeptide and a TGF-xcex2 receptor or receptor complex with a candidate pharmacological agent. In preferred embodiments of the foregoing methods, the step of forming a mixture further comprises adding a ligand which activates the TGF-xcex2 superfamily receptor or receptor complex.
In another aspect of the invention methods for reducing heteromeric Smad protein complex formation in a cell is provided. The methods include providing an antibody which selectively binds a phosphorylated pathway-restricted Smad polypeptide or a nucleic acid encoding the antibody, and contacting the cell with an amount of the antibody or the nucleic acid encoding the antibody sufficient to reduce formation of the heteromeric Smad protein complex in the cell. In certain embodiments, the pathway-restricted Smad polypeptide is selected from the group consisting of Smad1, Smad2, Smad3, Smad5, and Smad9. In other embodiments, the antibody selectively binds a pathway-restricted Smad polypeptide having at least one phosphorylated C-terminal serine residue selected from the group consisting of the second serine residue of SEQ ID NO:5, the third serine residue of SEQ ID NO:5 and the second and third serine residues of SEQ ID NO:5.
Use of the foregoing compounds in the preparation of a medicament also in provided.
These and other aspects of the invention will be described in further detail in connection with the detailed description of the invention.